The eccentric owner of Pekingese dog Tricki Woo was portrayed by Dame Diana Rigg in the first series, but the Doncaster-born star passed away last September.
Channel 5 and production company Playground have chosen Patricia Hodge as her replacement.
Hodge, 74, is the Cleethorpes-born daughter of Grimsby hoteliers and has appeared in Miranda and A Very English Scandal during a successful television and West End career.
Elsewhere the casting remains unchanged in the main roles, with Nicholas Ralph, Samuel West, Anna Madeley, Callum Woodhouse and Rachel Shenton all returning.
Another newcomer to the cast is Dorothy Atkinson, who played Jane Sutton in Call the Midwife. The 54-year-old's character is Diana Brompton, a love interest of Siegfied Farnon who catches his eye at a church dance. The Vicar of Dibley star James Fleet will also appear as Colonel Merrick, a local landowner.
Matthew Lewis, Cleo Sylvestre and Kriss Dosanjh will also reprise their roles from the first series.
Executive producer Sir Colin Callender CBE said: “We’ve been overwhelmed by the exceptional response to the first series and are very appreciative of the support from Channel 5, Masterpiece, All3Media and Screen Yorkshire who have enabled us to return to Darrowby so that audiences can once again enjoy the company of the beloved inhabitants of Skeldale House.
"We were deeply saddened by the passing of Dame Diana Rigg who will always be a treasured member of the All Creatures family. Mrs Pumphrey and her overly indulged Pekingese, Tricki Woo, are such iconic and cherished characters in Herriot’s world and we are delighted to welcome the wonderful Patricia Hodge who will join the company as Mrs Pumphrey for Series 2”.
There will be six episodes and a Christmas special in the new series.
Filming is currently taking place in Dales locations such as Grassington, Kettlewell and Arncliffe.